1. Field of the Invention
The present invention relates generally to a primer set for amplifying a target nucleic acid sequence, and a nucleic acid amplification method and mutation detection method that use the same.
2. Description of the Related Art
In, for example, genetic diagnoses of diseases and gene expression analysis, it is necessary to detect a target nucleic acid having a specific sequence. For the detection, fluorescence is used widely. Specifically, a known method uses a fluorescent substance whose fluorescence intensity increases with an increase in target nucleic acid sequence. A typical example of the fluorescent substance is a substance that intercalates into a double helix structure and emits fluorescence by irradiation with excitation light. When a target nucleic acid sequence is amplified using a primer labeled with such a fluorescent substance, for instance, the primer hybridizes to a target nucleic acid sequence of a template DNA to form a double strand, and thereby the fluorescent substance emits fluorescence. Therefore measurement of the fluorescence intensity of the fluorescent substance of the primer makes it possible to detect, for example, the occurrence or amount of amplification, or the presence or absence of a mutation to be detected in the target nucleic acid sequence.
However, there is a possibility that a conventional fluorescent substance may emit fluorescence, even when, for example, no double helix structure has been formed. Furthermore, for the purpose of quenching fluorescence of only a primer or probe labeled with a fluorescent substance, the method using fluorescence resonance energy transfer (FRET) is effective (for example, Tyagi, S., Kramer, F. R. (1996) Nat. Biotechnol. 14, 303-308; Nazarenko, I. A., Bhatnagar, S. K., Hohman, R. J. (1997) Nucleic Acids Res. 25, 2516-2521; Gelmini, S., Orlando, C., Sestini, R., Vona, G., Pinzani, P., Ruocco, L., Pazzagli, M. (1997) Clin. Chem. 43, 752-758; and Whitcombe, D., Theaker, J., Guy, S. P., Brown, T., Little, S. (1999) Nat. Biotechnol. 17, 804-807). However, it has problems in, for example, cost due to the introduction of two types of fluorescent dyes.
Thiazole orange, which is one type of cyanine dye, is known as a fluorescent dye whose fluorescence intensity increases through an interaction with DNA or RNA. There are examples in which a fluorescent probe was intended to be produced with thiazole orange being bonded to DNA by a covalent bond. However, it also emits strong fluorescence through an interaction with a single-stranded DNA containing a purine base (Biopolymers 1998, 46, 39-51). Accordingly, the increase in fluorescence intensity obtained when a double helix is formed is small, and therefore it cannot be considered as being successful (Analytica Chimica Acta 2002, 470, 57-70, and Chemistry—A European Journal 2006, 12, 2270-2281).
Furthermore, recently, the present inventors have reported an innovative method that allows a gene mutation to be detected easily and quickly by merely amplifying nucleic acid of a target nucleic acid sequence under an isothermal condition (Mitani Y., Lezhava A., Kawai Y., Kikuchi T., Oguchi-Katayama A., Kogo Y., Itoh M., Miyagi T. et al. 2007. “Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology.” Nat. Methods 4(3): 257-262; JP 3897926 B; and JP 3942627 B). Similarly in this method, a fluorescent substance as described above is used, and there are demands for a fluorescent substance that allows more effective detection.